The dramatic responses observed in patients with BRAF mutant melanoma treated with BRAF inhibitors reflect a major therapeutic breakthrough in melanoma. However, there are multiple resistance mechanisms that arise in patient tumors that limit the effectiveness of BRAF inhibitors. We have found that autophagy is activated in patient tumors and cell lines treated with BRAF inhibitors, and this therapy-associated autophagy protects melanoma cells and contributes to regrowth of tumors. Our previous work establishes that BRAF inhibitors activate autophagy by activating an early ER stress response which in turn gives rise to cytoprotective autophagy. In this proposal, the PI will work closely with the co-investigator, combining their respective expertise in autophagy, melanoma biology and treatment, and the ER stress response to elucidate the mechanistic underpinnings of the mutant BRAF-ER stress-autophagy signaling. Our hypothesis is that certain components of the ER stress pathway are critical for BRAF inhibitor-induced autophagy, and therefore could serve as novel targets for combinations regimens. Our strategy will be to first determine the molecular mechanism that links mutant BRAF with the ER stress response and autophagy (aim 1); then elucidate the biological effects of targeting specific components of the ER stress response or autophagy in BRAFi-induced cell death in 2D and 3D melanoma culture (aim 2); and to characterize the role of BRAFi-induced ER stress and autophagy in vivo using genetic and pharmacological inhibition of ER stress or autophagy in combination with BRAF inhibition (aim 3). The focus of these studies is on the development of a sophisticated understanding of the mechanistic links between these three pathways. The knowledge gained from these studies will increase our understanding about the fundamental biology of BRAF mutant melanoma, the interaction between mutant BRAF and the ER stress response, the role of cytoplasmic GRP78 in the response to targeted therapy, and the mechanisms by which the ER stress response regulates autophagy. Besides these fundamental advances, this project has translational potential because it will investigate these pathways in an enrolling trial of BRAF and autophagy inhibition in BRAF mutant melanoma patients, and it will identify potential new combinations that may be even more effective for future clinical trials.